Enhancing GABAergic Tonic Inhibition Reduces Seizure-Like Activity in the Neonatal Mouse Hippocampus and Neocortex.

Approximately one-third of neonatal seizures do not respond to first-line anticonvulsants, including phenobarbital, which enhances phasic inhibition. Whether enhancing tonic inhibition decreases seizure-like activity in the neonate when GABA is mainly depolarizing at this age is unknown. We evaluated if increasing tonic inhibition using THIP [4,5,6,7-tetrahydroisoxazolo(5,4-c)pyridin-3-ol, gaboxadol], a δ-subunit-selective GABAA receptor agonist, decreases seizure-like activity in neonatal C57BL/6J mice (postnatal day P5-8, both sexes) using acute brain slices. Whole-cell patch-clamp recordings showed that THIP enhanced GABAergic tonic inhibitory conductances in layer V neocortical and CA1 pyramidal neurons and increased their rheobase without altering sEPSC characteristics. Two-photon calcium imaging demonstrated that enhancing the activity of extrasynaptic GABAARs decreased neuronal firing in both brain regions. In the 4-aminopyridine and the low-Mg2+ model of pharmacoresistant seizures, THIP reduced epileptiform activity in the neocortex and CA1 hippocampal region of neonatal and adult brain slices in a dose-dependent manner. We conclude that neocortical layer V and CA1 pyramidal neurons have tonic inhibitory conductances, and when enhanced, they reduce neuronal firing and decrease seizure-like activity. Therefore, augmenting tonic inhibition could be a viable approach for treating neonatal seizures.

A Novel CCK Receptor GPR173 Mediates Potentiation of GABAergic Inhibition.

Cholecystokinin (CCK) enables excitatory circuit long-term potentiation (LTP). Here, we investigated its involvement in the enhancement of inhibitory synapses. Activation of GABA neurons suppressed neuronal responses in the neocortex to a forthcoming auditory stimulus in mice of both sexes. High-frequency laser stimulation (HFLS) of GABAergic neurons potentiated this suppression. HFLS of CCK interneurons could induce the LTP of their inhibition toward pyramidal neurons. This potentiation was abolished in CCK knock-out mice but intact in mice with both CCK1R and 2R knockout of both sexes. Next, we combined bioinformatics analysis, multiple unbiased cell-based assays, and histology examinations to identify a novel CCK receptor, GPR173. We propose GPR173 as CCK3R, which mediates the relationship between cortical CCK interneuron signaling and inhibitory LTP in the mice of either sex. Thus, GPR173 might represent a promising therapeutic target for brain disorders related to excitation and inhibition imbalance in the cortex.SIGNIFICANCE STATEMENT CCK, the most abundant and widely distributed neuropeptide in the CNS, colocalizes with many neurotransmitters and modulators. GABA is one of the important inhibitory neurotransmitters, and much evidence shows that CCK may be involved in modulating GABA signaling in many brain areas. However, the role of CCK-GABA neurons in the cortical microcircuits is still unclear. We identified a novel CCK receptor, GPR173, localized in the CCK-GABA synapses and mediated the enhancement of the GABA inhibition effect, which might represent a promising therapeutic target for brain disorders related to excitation and inhibition imbalance in the cortex.

Stringent specificity in the construction of a GABAergic presynaptic inhibitory circuit.

GABAergic interneurons are key elements in neural coding, but the mechanisms that assemble inhibitory circuits remain unclear. In the spinal cord, the transfer of sensory signals to motor neurons is filtered by GABAergic interneurons that act presynaptically to inhibit sensory transmitter release and postsynaptically to inhibit motor neuron excitability. We show here that the connectivity and synaptic differentiation of GABAergic interneurons that mediate presynaptic inhibition is directed by their sensory targets. In the absence of sensory terminals these GABAergic neurons shun other available targets, fail to undergo presynaptic differentiation, and withdraw axons from the ventral spinal cord. A sensory-specific source of brain derived neurotrophic factor induces synaptic expression of the GABA synthetic enzyme GAD65--a defining biochemical feature of this set of interneurons. The organization of a GABAergic circuit that mediates presynaptic inhibition in the mammalian CNS is therefore controlled by a stringent program of sensory recognition and signaling.

Carbachol increases locus coeruleus activation by targeting noradrenergic neurons, inhibitory interneurons and inhibitory synaptic transmission.

The locus coeruleus (LC) consists of noradrenergic (NA) neurons and plays an important role in controlling behaviours. Although much of the knowledge regarding LC functions comes from studying behavioural outcomes upon administration of muscarinic acetylcholine receptor (mAChR) agonists into the nucleus, the exact mechanisms remain unclear. Here, we report that the application of carbachol (CCh), an mAChR agonist, increased the spontaneous action potentials (sAPs) of both LC-NA neurons and local inhibitory interneurons (LC I-INs) in acute brain slices by activating M1/M3 mAChRs (m1/3 AChRs). Optogenetic activation of LC I-INs evoked inhibitory postsynaptic currents (IPSCs) in LC-NA neurons that were mediated by γ-aminobutyric acid type A (GABAA ) and glycine receptors, and CCh application decreased the IPSC amplitude through a presynaptic mechanism by activating M4 mAChRs (m4 AChRs). LC-NA neurons also exhibited spontaneous phasic-like activity (sPLA); CCh application increased the incidence of this activity. This effect of CCh application was not observed with blockade of GABAA and glycine receptors, suggesting that the sPLA enhancement occurred likely because of the decreased synaptic transmission of LC I-INs onto LC-NA neurons by the m4 AChR activation and/or increased spiking rate of LC I-INs by the m1/3 AChR activation, which could lead to fatigue of the synaptic transmission. In conclusion, we report that CCh application, while inhibiting their synaptic transmission, increases sAP rates of LC-NA neurons and LC I-INs. Collectively, these effects provide insight into the cellular mechanisms underlying the behaviour modulations following the administration of muscarinic receptor agonists into the LC reported by the previous studies.

Excitatory/inhibitory balance in epilepsies and neurodevelopmental disorders: Depolarizing γ-aminobutyric acid as a common mechanism.

Epilepsy is one of the most common neurological disorders. Although many factors contribute to epileptogenesis, seizure generation is mostly linked to hyperexcitability due to alterations in excitatory/inhibitory (E/I) balance. The common hypothesis is that reduced inhibition, increased excitation, or both contribute to the etiology of epilepsy. Increasing evidence shows that this view is oversimplistic, and that increased inhibition through depolarizing γ-aminobutyric acid (GABA) similarly contributes to epileptogenisis. In early development, GABA signaling is depolarizing, inducing outward Cl- currents due to high intracellular Cl- concentrations. During maturation, the mechanisms of GABA action shift from depolarizing to hyperpolarizing, a critical event during brain development. Altered timing of this shift is associated with both neurodevelopmental disorders and epilepsy. Here, we consider the different ways that depolarizing GABA contributes to altered E/I balance and epileptogenesis, and discuss that alterations in depolarizing GABA could be a common denominator underlying seizure generation in neurodevelopmental disorders and epilepsies.

GABAergic Signaling during Spinal Cord Stimulation Reduces Cardiac Arrhythmias in a Porcine Model.

BACKGROUND: Neuraxial modulation, including spinal cord stimulation, reduces cardiac sympathoexcitation and ventricular arrhythmogenesis. There is an incomplete understanding of the molecular mechanisms through which spinal cord stimulation modulates cardiospinal neural pathways. The authors hypothesize that spinal cord stimulation reduces myocardial ischemia-reperfusion-induced sympathetic excitation and ventricular arrhythmias through γ-aminobutyric acid (GABA)-mediated pathways in the thoracic spinal cord. METHODS: Yorkshire pigs were randomized to control (n = 11), ischemia-reperfusion (n = 16), ischemia-reperfusion plus spinal cord stimulation (n = 17), ischemia-reperfusion plus spinal cord stimulation plus γ-aminobutyric acid type A (GABAA) or γ-aminobutyric acid type B (GABAB) receptor antagonist (GABAA, n = 8; GABAB, n = 8), and ischemia-reperfusion plus GABA transaminase inhibitor (GABAculine, n = 8). A four-pole spinal cord stimulation lead was placed epidurally (T1 to T4). GABA modulating pharmacologic agents were administered intrathecally. Spinal cord stimulation at 50 Hz was applied 30 min before ischemia. A 56-electrode epicardial mesh was used for high-resolution electrophysiologic recordings, including activation recovery intervals and ventricular arrhythmia scores. Immunohistochemistry and Western blots were performed to measure GABA receptor expression in the thoracic spinal cord. RESULTS: Cardiac ischemia led to myocardial sympathoexcitation with reduction in activation recovery interval (mean ± SD, -42 ± 11%), which was attenuated by spinal cord stimulation (-21 ± 17%, P = 0.001). GABAA and GABAB receptor antagonists abolished spinal cord stimulation attenuation of sympathoexcitation (GABAA, -9.7 ± 9.7%, P = 0.043 vs. ischemia-reperfusion plus spinal cord stimulation; GABAB, -13 ± 14%, P = 0.012 vs. ischemia-reperfusion plus spinal cord stimulation), while GABAculine alone caused a therapeutic effect similar to spinal cord stimulation (-4.1 ± 3.7%, P = 0.038 vs. ischemia-reperfusion). The ventricular arrhythmia score supported these findings. Spinal cord stimulation during ischemia-reperfusion increased GABAA receptor expression with no change in GABAB receptor expression. CONCLUSIONS: Thoracic spinal cord stimulation reduces ischemia-reperfusion-induced sympathoexcitation and ventricular arrhythmias through activation of GABA signaling pathways. These data support the hypothesis that spinal cord stimulation-induced release of GABA activates inhibitory interneurons to decrease primary afferent signaling from superficial dorsal horn to sympathetic output neurons in the intermediolateral nucleus.

Non-monotonic effects of GABAergic synaptic inputs on neuronal firing.

GABA is generally known as the principal inhibitory neurotransmitter in the nervous system, usually acting by hyperpolarizing membrane potential. However, GABAergic currents sometimes exhibit non-inhibitory effects, depending on the brain region, developmental stage or pathological condition. Here, we investigate the diverse effects of GABA on the firing rate of several single neuron models, using both analytical calculations and numerical simulations. We find that GABAergic synaptic conductance and output firing rate exhibit three qualitatively different regimes as a function of GABA reversal potential, EGABA: monotonically decreasing for sufficiently low EGABA (inhibitory), monotonically increasing for EGABA above firing threshold (excitatory); and a non-monotonic region for intermediate values of EGABA. In the non-monotonic regime, small GABA conductances have an excitatory effect while large GABA conductances show an inhibitory effect. We provide a phase diagram of different GABAergic effects as a function of GABA reversal potential and glutamate conductance. We find that noisy inputs increase the range of EGABA for which the non-monotonic effect can be observed. We also construct a micro-circuit model of striatum to explain observed effects of GABAergic fast spiking interneurons on spiny projection neurons, including non-monotonicity, as well as the heterogeneity of the effects. Our work provides a mechanistic explanation of paradoxical effects of GABAergic synaptic inputs, with implications for understanding the effects of GABA in neural computation and development.

Basal Ganglia Output Has a Permissive Non-Driving Role in a Signaled Locomotor Action Mediated by the Midbrain.

The basal ganglia are important for movement and reinforcement learning. Using mice of either sex, we found that the main basal ganglia GABAergic output in the midbrain, the substantia nigra pars reticulata (SNr), shows movement-related neural activity during the expression of a negatively reinforced signaled locomotor action known as signaled active avoidance; this action involves mice moving away during a warning signal to avoid a threat. In particular, many SNr neurons deactivate during active avoidance responses. However, whether SNr deactivation has an essential role driving or regulating active avoidance responses is unknown. We found that optogenetic excitation of SNr or striatal GABAergic fibers that project to an area in the pedunculopontine tegmentum (PPT) within the midbrain locomotor region abolishes signaled active avoidance responses, while optogenetic inhibition of SNr cells (mimicking the SNr deactivation observed during an active avoidance behavior) serves as an effective conditioned stimulus signal to drive avoidance responses by disinhibiting PPT neurons. However, preclusion of SNr deactivation, or direct inhibition of SNr fibers in the PPT, does not impair the expression of signaled active avoidance, indicating that SNr output does not drive the expression of a signaled locomotor action mediated by the midbrain. Consistent with a permissive regulatory role, SNr output provides information about the state of the ongoing action to downstream structures that mediate the action.SIGNIFICANCE STATEMENT During signaled active avoidance behavior, subjects move away to avoid a threat when directed by an innocuous sensory stimulus. Excitation of GABAergic cells in the substantia nigra pars reticulata (SNr), the main output of the basal ganglia, blocks signaled active avoidance, while inhibition of SNr cells is an effective stimulus to drive active avoidance. Interestingly, many SNr cells inhibit their firing during active avoidance responses, suggesting that SNr inhibition could be driving avoidance responses by disinhibiting downstream areas. However, interfering with the modulation of SNr cells does not impair the behavior. Thus, SNr may regulate the active avoidance movement in downstream areas that mediate the behavior, but does not drive it.

Enhancing GABAergic Tone in the Rostral Nucleus of the Solitary Tract Reconfigures Sensorimotor Neural Activity.

Recent work has shown that most cells in the rostral, gustatory portion of the nucleus tractus solitarius (rNTS) in awake, freely licking rats show lick-related firing. However, the relationship between taste-related and lick-related activity in rNTS remains unclear. Here, we tested whether GABA-derived inhibitory activity regulates the balance of lick- and taste-driven neuronal activity. Combinatorial viral tools were used to restrict the expression of channelrhodopsin 2-enhanced yellow fluorescent protein to GAD1+ GABAergic neurons. Viral infusions were bilateral in rNTS. A fiber-optic fiber attached to a bundle of drivable microwires was later implanted into the rNTS. After recovery, water-deprived rats were presented with taste stimuli in an experimental chamber. Trials were five consecutive taste licks [NaCl, KCl, NH4Cl, sucrose, monosodium glutamate/inosine-5'-monophosphate, citric acid, quinine, or artificial saliva (AS)] separated by five AS rinse licks on a variable ratio 5 schedule. Each taste lick triggered a 1 s train of laser light (25 Hz; 473 nm; 8-10 mW) in a random half of the trials. In all, 113 cells were recorded in the rNTS, 50 cells responded to one or more taste stimuli without GABA enhancement. Selective changes in response magnitude (spike count) within cells shifted across-unit patterns but preserved interstimulus relationships. Cells where enhanced GABAergic tone increased lick coherence conveyed more information distinguishing basic taste qualities and different salts than other cells. In addition, GABA activation significantly amplified the amount of information that discriminated palatable versus unpalatable tastants. By dynamically regulating lick coherence and remodeling the across-unit response patterns to taste, enhancing GABAergic tone in rNTS reconfigures the neural activity reflecting sensation and movement.

Flexible versus Fixed Spatial Self-Ordered Response Sequencing: Effects of Inactivation and Neurochemical Modulation of Ventrolateral Prefrontal Cortex.

Previously, studies using human neuroimaging and excitotoxic lesions in non-human primate have demonstrated an important role of ventrolateral prefrontal cortex (vlPFC) in higher order cognitive functions such as cognitive flexibility and the planning of behavioral sequences. In the present experiments, we tested effects on performance of temporary inactivation (using GABA receptor agonists) and dopamine (DA) D2 and 5-HT2A-receptor (R) blockade of vlPFC via local intracerebral infusions in the marmoset. We trained common marmosets to perform spatial self-ordered sequencing tasks in which one cohort of animals performed two and three response sequences on a continuously varying spatial array of response options on a touch-sensitive screen. Inactivation of vlPFC produced a marked disruption of accuracy of sequencing which also exhibited significant error perseveration. There were somewhat contrasting effects of D2 and 5-HT2A-R blockade, with the former producing error perseveration on incorrect trials, though not significantly impairing accuracy overall, and the latter significantly impairing accuracy but not error perseveration. A second cohort of marmosets were directly compared on performance of fixed versus variable spatial arrays. Inactivation of vlPFC again impaired self-ordered sequencing, but only with varying, and not fixed spatial arrays, the latter leading to the consistent use of fewer, preferred sequences. These findings add to evidence that vlPFC is implicated in goal-directed behavior that requires higher-order response heuristics that can be applied flexibly over different (variable), as compared with fixed stimulus exemplars. They also show that dopaminergic and serotonergic chemomodulation has distinctive effects on such performance.SIGNIFICANCE STATEMENT This investigation employing local intracerebral infusions to inactivate the lateral prefrontal cortex (PFC) of the New World marmoset reveals the important role of this region in self-ordered response sequencing in variable but not fixed spatial arrays. These novel findings emphasize the higher order functions of this region, contributing to cognitive flexibility and planning of goal directed behavior. The investigation also reports for the first time somewhat contrasting neuromodulatory deficits produced by infusions of dopamine (DA) D2 and 5-HT2A receptor (R) antagonists into the same region, of possible significance for understanding cognitive deficits produced by anti-psychotic drugs.

Photoreceptive Ganglion Cells Drive Circuits for Local Inhibition in the Mouse Retina.

Intrinsically photosensitive retinal ganglion cells (ipRGCs) exhibit melanopsin-dependent light responses that persist in the absence of rod and cone photoreceptor-mediated input. In addition to signaling anterogradely to the brain, ipRGCs signal retrogradely to intraretinal circuitry via gap junction-mediated electrical synapses with amacrine cells (ACs). However, the targets and functions of these intraretinal signals remain largely unknown. Here, in mice of both sexes, we identify circuitry that enables M5 ipRGCs to locally inhibit retinal neurons via electrical synapses with a nonspiking GABAergic AC. During pharmacological blockade of rod- and cone-mediated input, whole-cell recordings of corticotropin-releasing hormone-expressing (CRH+) ACs reveal persistent visual responses that require both melanopsin expression and gap junctions. In the developing retina, ipRGC-mediated input to CRH+ ACs is weak or absent before eye opening, indicating a primary role for this input in the mature retina (i.e., in parallel with rod- and cone-mediated input). Among several ipRGC types, only M5 ipRGCs exhibit consistent anatomical and physiological coupling to CRH+ ACs. Optogenetic stimulation of local CRH+ ACs directly drives IPSCs in M4 and M5, but not M1-M3, ipRGCs. CRH+ ACs also inhibit M2 ipRGC-coupled spiking ACs, demonstrating direct interaction between discrete networks of ipRGC-coupled interneurons. Together, these results demonstrate a functional role for electrical synapses in translating ipRGC activity into feedforward and feedback inhibition of local retinal circuits.SIGNIFICANCE STATEMENT Melanopsin directly generates light responses in intrinsically photosensitive retinal ganglion cells (ipRGCs). Through gap junction-mediated electrical synapses with retinal interneurons, these uniquely photoreceptive RGCs may also influence the activity and output of neuronal circuits within the retina. Here, we identified and studied an electrical synaptic circuit that, in principle, could couple ipRGC activity to the chemical output of an identified retinal interneuron. Specifically, we found that M5 ipRGCs form electrical synapses with corticotropin-releasing hormone-expressing amacrine cells, which locally release GABA to inhibit specific RGC types. Thus, ipRGCs are poised to influence the output of diverse retinal circuits via electrical synapses with interneurons.

Increased GABA transmission to GnRH neurons after intrahippocampal kainic acid injection in mice is sex-specific and associated with estrous cycle disruption.

Patients with epilepsy develop reproductive endocrine comorbidities at a rate higher than that of the general population. Clinical studies have identified disrupted luteinizing hormone (LH) release patterns in patients of both sexes, suggesting potential epilepsy-associated changes in hypothalamic gonadotropin-releasing hormone (GnRH) neuron function. In previous work, we found that GnRH neuron firing is increased in diestrous females and males in the intrahippocampal kainic acid (IHKA) mouse model of temporal lobe epilepsy. Notably, GABAA receptor activation is depolarizing in adult GnRH neurons. Therefore, here we tested the hypothesis that increased GnRH neuron firing in IHKA mice is associated with increased GABAergic drive to GnRH neurons. When ionotropic glutamate receptors (iGluRs) were blocked to isolate GABAergic postsynaptic currents (PSCs), no differences in PSC frequency were seen between GnRH neurons from control and IHKA diestrous females. In the absence of iGluR blockade, however, GABA PSC frequency was increased in GnRH neurons from IHKA females with disrupted estrous cycles, but not saline-injected controls nor IHKA females without estrous cycle disruption. GABA PSC amplitude was also increased in IHKA females with disrupted estrous cycles. These findings suggest the presence of an iGluR-dependent increase in feed-forward GABAergic transmission to GnRH neurons specific to IHKA females with comorbid cycle disruption. In males, GABA PSC frequency and amplitude were unchanged but PSC duration was reduced. Together, these findings suggest that increased GABA transmission helps drive elevated firing in IHKA females on diestrus and indicate the presence of a sex-specific hypothalamic mechanism underlying reproductive endocrine dysfunction in IHKA mice.

GABA-ergic agents modulated the effects of histamine on the behaviour of male mice in the elevated plus maze test.

NEW FINDINGS: What is the central question of this study? Is there an interaction between histamine and the GABAergic system in modulation of anxiety in mice? What is the main finding and its importance? There is a synergistic anxiogenic effect between histamine and bicuculline in mice. This effect may be due to a direct or an indirect effect of the histaminergic system on the GABAergic system. ABSTRACT: It has been documented that both histaminergic and GABAergic systems participate in the neurobiology of anxiety behaviour. In the current research, we investigated the effects of the histaminergic system and GABAA receptor agents on anxiety-related behaviours and their interaction using the elevated plus maze test in mice. Intraperitoneal (i.p.) administration of muscimol (0.12 and 0.25 mg/kg) increased the open arm time (OAT) (P < 0.001) without affecting the open arm entries (OAE) and locomotor activity, showing an anxiolytic effect. i.p. injection of bicuculline (0.5 and 1 mg/kg) decreased OAT (P < 0.001) but not OAE and locomotor activity, suggesting an anxiogenic behaviour. Intracerebroventricular (i.c.v.) microinjection of histamine (2.5 and 5 µg/mouse) resulted in a decline in OAT (P < 0.001) but not OAE and locomotor activity, indicating an anxiogenic response. Co-administration of histamine with GABAergic agents, muscimol (0.06 mg/kg; i.p.) and bicuculline (0.25 mg/kg; i.p.), decreased (P < 0.001) and increased (P < 0.05), respectively, the anxiogenic-like response to the effective dose (5 µg/mouse; i.c.v.) of histamine. In addition, co-treatment of effective doses of histamine (2.5 and 5 µg/mouse;i.c.v.) with an effective dose of muscimol (0.12 mg/kg; i.p.) and a non-effective dose of bicuculline (0.25 mg/kg; i.p.) significantly decreased OAT (P < 0.001), suggesting a likely interaction between the histaminergic and GABAergic systems in the regulation of anxiety. The results demonstrated a synergistic anxiogenic-like effect between histamine and bicuculline in mice. In conclusion, our results present an interaction between the histaminergic and GABAergic systems in anxiolytic/anxiogenic-like behaviours in the elevated plus maze test.

Consequences: Bench to home.

Seizure clusters (also referred to as acute repetitive seizures) consist of several seizures interspersed with brief interictal periods. Seizure clusters can break down γ-aminobutyric acidergic (GABAergic) inhibition of dentate granule cells, leading to hyperactivation. Functional changes to GABAA receptors, which play a vital neuroinhibitory role, can include altered GABAA receptor subunit trafficking and cellular localization, intracellular chloride accumulation, and dysregulation of proteins critical to chloride homeostasis. A reduction in neuroinhibition and potentiation of excitatory neurotransmission in CA1 pyramidal neurons represent pathological mechanisms that underlie seizure clusters. Benzodiazepines are well-established treatments for seizure clusters; however, there remain barriers to appropriate care. At the clinical level, there is variability in seizure cluster definitions, such as the number and/or type of seizures associated with a cluster as well as the interictal duration between seizures. This can lead to delays in diagnosis and timely treatment. There are gaps in understanding between clinicians, their patients, and caregivers regarding acute treatment for seizure clusters, such as the use of rescue medications and emergency services. This lack of consensus to define seizure clusters in addition to a lack of education for appropriate treatment can affect quality of life for patients and place a greater burden on patient families and caregivers. For patients with seizure clusters, the sense of unpredictability can lead to continuous traumatic stress, during which patients and families live with a heightened level of anxiety. Clinicians can affect patient quality of life and clinical outcomes through improved seizure cluster education and treatment, such as the development and implementation of a personalized seizure action plan as well as prescriptions for suitable rescue medications indicated for seizure clusters and instructions for their proper use. In all, the combination of targeted therapy along with patient education and support can improve quality of life.

Novel hybrid action of GABA mediates inhibitory feedback in the mammalian retina.

The stream of visual information sent from photoreceptors to second-order bipolar cells is intercepted by laterally interacting horizontal cells that generate feedback to optimize and improve the efficiency of signal transmission. The mechanisms underlying the regulation of graded photoreceptor synaptic output in this nonspiking network have remained elusive. Here, we analyze with patch clamp recording the novel mechanisms by which horizontal cells control pH in the synaptic cleft to modulate photoreceptor neurotransmitter release. First, we show that mammalian horizontal cells respond to their own GABA release and that the results of this autaptic action affect cone voltage-gated Ca2+ channel (CaV channel) gating through changes in pH. As a proof-of-principle, we demonstrate that chemogenetic manipulation of horizontal cells with exogenous anion channel expression mimics GABA-mediated cone CaV channel inhibition. Activation of these GABA receptor anion channels can depolarize horizontal cells and increase cleft acidity via Na+/H+ exchanger (NHE) proton extrusion, which results in inhibition of cone CaV channels. This action is effectively counteracted when horizontal cells are sufficiently hyperpolarized by increased GABA receptor (GABAR)-mediated HCO3- efflux, alkalinizing the cleft and disinhibiting cone CaV channels. This demonstrates how hybrid actions of GABA operate in parallel to effect voltage-dependent pH changes, a novel mechanism for regulating synaptic output.

Modelling the spatial and temporal constrains of the GABAergic influence on neuronal excitability.

GABA (γ-amino butyric acid) is an inhibitory neurotransmitter in the adult brain that can mediate depolarizing responses during development or after neuropathological insults. Under which conditions GABAergic membrane depolarizations are sufficient to impose excitatory effects is hard to predict, as shunting inhibition and GABAergic effects on spatiotemporal filtering of excitatory inputs must be considered. To evaluate at which reversal potential a net excitatory effect was imposed by GABA (EGABAThr), we performed a detailed in-silico study using simple neuronal topologies and distinct spatiotemporal relations between GABAergic and glutamatergic inputs. These simulations revealed for GABAergic synapses located at the soma an EGABAThr close to action potential threshold (EAPThr), while with increasing dendritic distance EGABAThr shifted to positive values. The impact of GABA on AMPA-mediated inputs revealed a complex temporal and spatial dependency. EGABAThr depends on the temporal relation between GABA and AMPA inputs, with a striking negative shift in EGABAThr for AMPA inputs appearing after the GABA input. The spatial dependency between GABA and AMPA inputs revealed a complex profile, with EGABAThr being shifted to values negative to EAPThr for AMPA synapses located proximally to the GABA input, while for distally located AMPA synapses the dendritic distance had only a minor effect on EGABAThr. For tonic GABAergic conductances EGABAThr was negative to EAPThr over a wide range of gGABAtonic values. In summary, these results demonstrate that for several physiologically relevant situations EGABAThr is negative to EAPThr, suggesting that depolarizing GABAergic responses can mediate excitatory effects even if EGABA did not reach EAPThr.

Kresimir Krnjevic (1927-2021) and GABAergic inhibition: a lifetime dedication.

After over seven decades of neuroscience research, it is now well established that γ-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the brain. In this paper dedicated to Kresimir Krnjevic (1927-2021), a pioneer and leader in neuroscience, we briefly highlight the fundamental contributions he made in identifying GABA as an inhibitory neurotransmitter in the brain and our personal interactions with him. Of note, between 1972 and 1978 Dr. Krnjevic was a highly reputed Chief Editor of the Canadian Journal of Physiology and Pharmacology.

Excitatory-Inhibitory Synaptic Coupling in Avian Nucleus Magnocellularis.

The activity of neurons is determined by the balance between their excitatory and inhibitory synaptic inputs. Neurons in the avian nucleus magnocellularis (NM) integrate monosynaptic excitatory and polysynaptic inhibitory inputs from the auditory nerve, and transmit phase-locked output to higher auditory centers. The excitatory input is graded tonotopically, such that neurons tuned to higher frequency receive fewer, but larger, axon terminals. However, it remains unknown how the balance between excitatory and inhibitory inputs is determined in NM. We here examined synaptic and spike responses of NM neurons during stimulation of the auditory nerve in thick brain slices of chicken of both sexes, and found that the excitatory-inhibitory balance varied according to tonotopic region, ensuring reliable spike output across frequencies. Auditory nerve stimulation elicited IPSCs in NM neurons regardless of tonotopic region, but the dependence of IPSCs on intensity varied in a systematic way. In neurons tuned to low frequency, IPSCs appeared and increased in parallel with EPSCs with elevation of intensity, which expanded dynamic range by preventing saturation of spike generation. On the other hand, in neurons tuned to higher frequency, IPSCs were smaller than EPSCs and had higher thresholds for activation, thus facilitating high-fidelity transmission. Computer simulation confirmed that these differences in inhibitory input were optimally matched to the patterns of excitatory input, and enabled appropriate level of neuronal output for wide intensity and frequency ranges of sound in the auditory system.SIGNIFICANCE STATEMENT Neurons in nucleus magnocellularis encode timing information of sound across wide intensity ranges by integrating excitatory and inhibitory synaptic inputs from the auditory nerve, but underlying synaptic mechanisms of this integration are not fully understood. We here show that the excitatory-inhibitory relationship was expressed differentially at each tonotopic region; the relationship was linear in neurons tuned to low-frequency, expanding dynamic range by preventing saturation of spike generation; by contrast inhibitory input remained much smaller than excitatory input in neurons tuned to higher frequency, thus ensuring high-fidelity transmission. The tonotopic regulation of excitatory and inhibitory input optimized the output across frequencies and intensities, playing a fundamental role in the timing coding pathway in the auditory system.

Discharge and Role of GABA Pontomesencephalic Neurons in Cortical Activity and Sleep-Wake States Examined by Optogenetics and Juxtacellular Recordings in Mice.

The cholinergic neurons in the pontomesencephalic tegmentum have been shown to discharge in association with and promote cortical activation during active or attentive waking and paradoxical or rapid eye movement sleep. However, GABA neurons lie intermingled with the cholinergic neurons and may contribute to or oppose this activity and role. Here we investigated in vitro and in vivo the properties, activities, and role of GABA neurons within the laterodorsal tegmental and sublaterodorsal tegmental nuclei (LDT/SubLDT) using male and female transgenic mice expressing channelrhodopsin-(ChR2)-EYFP in vesicular GABA transporter (VGAT)-expressing neurons. Presumed GABA (pGABA) neurons were identified by response to photostimulation and verified by immunohistochemical staining following juxtacellular labeling in vivo pGABA neurons were found to be fast-firing neurons with the capacity to burst when depolarized from a hyperpolarized membrane potential. When stimulated in vivo in urethane-anesthetized or unanesthetized mice, the pGABA neurons fired repetitively at relatively fast rates (∼40 Hz) during a continuous light pulse or phasically in bursts (>100 Hz) when driven by rhythmic light pulses at theta (4 or 8 Hz) frequencies. pNon-GABA, which likely included cholinergic, neurons were inhibited during each light pulse to discharge rhythmically in antiphase to the pGABA neurons. The reciprocal rhythmic bursting by the pGABA and pNon-GABA neurons drove rhythmic theta activity in the EEG. Such phasic bursting by GABA neurons also occurred in WT mice in association with theta activity during attentive waking and paradoxical sleep.SIGNIFICANCE STATEMENT Neurons in the pontomesencephalic tegmentum, particularly cholinergic neurons, play an important role in cortical activation, which occurs during active or attentive waking and paradoxical or rapid eye movement sleep. Yet the cholinergic neurons lie intermingled with GABA neurons, which could play a similar or opposing role. Optogenetic stimulation and recording of these GABA neurons in mice revealed that they can discharge in rhythmic bursts at theta frequencies and drive theta activity in limbic cortex. Such phasic burst firing also occurs during natural attentive waking and paradoxical sleep in association with theta activity and could serve to enhance sensory-motor processing and memory consolidation during these states.

Anandamide Signaling Augmentation Rescues Amygdala Synaptic Function and Comorbid Emotional Alterations in a Model of Epilepsy.

Epilepsy is often associated with emotional disturbances and the endocannabinoid (eCB) system tunes synaptic transmission in brain regions regulating emotional behavior. Thus, persistent alteration of eCB signaling after repeated seizures may contribute to the development of epilepsy-related emotional disorders. Here we report that repeatedly eliciting seizures (kindling) in the amygdala caused a long-term increase in anxiety and impaired fear memory retention, which was paralleled by an imbalance in GABA/glutamate presynaptic activity and alteration of synaptic plasticity in the basolateral amygdala (BLA), in male rats. Anandamide (AEA) content was downregulated after repeated seizures, and pharmacological enhancement of AEA signaling rescued seizure-induced anxiety by restoring the tonic control of the eCB signaling over glutamatergic transmission. Moreover, AEA signaling augmentation also rescued the seizure-induced alterations of fear memory by restoring the phasic control of eCB signaling over GABAergic activity and plasticity in the BLA. These results indicate that modulation of AEA signaling represents a potential and promising target for the treatment of comorbid emotional dysfunction associated with epilepsy.SIGNIFICANCE STATEMENT Epilepsy is a heterogeneous neurologic disorder commonly associated with comorbid emotional alterations. However, the management of epilepsy is usually restricted to the control of seizures. The endocannabinoid (eCB) system, particularly anandamide (AEA) signaling, controls neuronal excitability and seizure expression and regulates emotional behavior. We found that repeated seizures cause an allostatic maladaptation of AEA signaling in the amygdala that drives emotional alterations. Boosting AEA signaling through inhibition of its degradative enzyme, fatty acid amide hydrolase (FAAH), restored both synaptic and behavioral alterations. FAAH inhibitors dampen seizure activity in animal models and are used in clinical studies to treat the negative consequences associated with stress. Thereby, they are accessible and can be clinically evaluated to treat both seizures and comorbid conditions associated with epilepsy.